1. Field of the Invention
This invention relates to long-chain acyl-coenzyme-A synthetase (abbreviated throughout the specification and claims as acyl-CoA synthetase, EC. 6.2.1.3), and a process for the purification of the enzyme.
2. Description of the Prior Art
The acyl-CoA synthetase is an important enzyme participating in the first stage of oxidation of fatty acids in the living body and catalyzes the following reaction: EQU RCOOH+CoA+ATP.fwdarw.RCOCoA+AMP+Pyrophosphoric Acid (1)
(In the above formula (1), R represents a saturated or unsaturated alkyl.)
This enzyme is known to occur in the rat liver, various bacteria and yeasts, for example, Escherichia coli and bacilli. Heretofore, many attempts have been made to purify the enzyme, but none of them have been successful in isolating the enzyme as a pure preparation because of its tendency to bind to the membranes as well as its instability.
For example, D. Samuel et al in the European Journal of Biochemistry, Vol. 12, pages 576-582 (1970) made purification of acyl-CoA synthetase from Escherichia coli (hereinafter referred to as E. coli), but they failed to purify the enzyme as an entirely homogeneous protein, the purified product having a low specific activity per milligram of protein.
Purification of the long-chain acyl-CoA synthetase from microsomes of the rat liver was also attempted by J. Bar-Tana et al in the Biochemical Journal, Vol. 122, pages 353-362 (1971), but, the homogeneity of the purified enzyme was not sufficient from the electrophoretic data and the specific activity thereof was also low.
The inventors previously found in the course of their study on acyl-CoA synthetase from microorganisms that the enzyme included two distinct enzymes, acyl-CoA synthetases I and II wherein acyl-CoA synthetase I participated in the systems for direct incorporation of fatty acids into lipids and acyl-CoA synthetase II participated in the .beta.-oxidation systems of fatty acids [Mishina et al, European Journal of Biochemistry, Vol. 82, pages 347-354 (1978)].
Among these, acyl-CoA synthetase is the enzyme which the present invention concerns. (In the following description, by the expression "the enzyme" is meant acyl-CoA synthetase I unless otherwise specified.)
Upon extensive investigation on the purification of longchain acyl-CoA synthetase I of microorganism origin, the inventors have succeeded in isolating the enzyme having a significantly high specific activity and homogeneous in the electrophoresis.